1. Field of the Invention
The present invention relates to a method for producing an organic acid such as succinic acid using a bacterium.
2. Brief Description of the Related Art
For the production of non-amino organic acids, including succinic acid, by fermentation, anaerobic bacteria are typically used, including bacteria belonging to the genus Anaerobiospirillum or Actinobacillus (U.S. Pat. Nos. 5,142,833, 5,504,004, International Journal of Systematic Bacteriology (1999), 49, 207-216). Although such anaerobic bacteria provide high product yields, many nutrients are required for their growth, and therefore it is necessary to add large amounts of organic nitrogen sources, such as corn steep liquor (CSL), into the culture medium. The addition of large amounts of sources of organic nitrogen results in not only an increase in cost for the culture medium, but also an increase in the purification cost for isolating the product, and therefore it is not economical.
In addition, methods are known in which aerobic bacteria such as coryneform bacteria are cultured once under aerobic conditions to proliferate the bacterial cells, then the bacteria are harvested and washed. As a result, a non-amino organic acid is produced without having to supply oxygen (Japanese Patent Laid-open (KOKAI) Nos. 11-113588 and 11-196888). These methods are economical, since smaller amounts of organic nitrogen can be added, and sufficient growth of the bacteria can be obtained in a simple culture medium. However, there is still room for improvement in terms of production amounts, concentration, and production rate per cell of the target organic acids, and the like. Furthermore, the production process could be simplified.
Escherichia coli is a facultative anaerobic gram negative bacterium, and similar to when using coryneform bacteria, methods are known for producing a non-amino organic acid by culturing the bacteria once under aerobic conditions to allow for bacterial growth, and then culturing again in the absence of oxygen to anaerobically produce the non-amino organic acid (Journal of Industrial Microbiology and Biotechnology (2002), 28 (6), 325-332). Alternatively, the bacteria can be aerobically cultured to aerobically produce the non-amino organic acid (U.S. Patent Published Application No. 20050170482). However, since Escherichia coli is a gram-negative bacterium, it is vulnerable to osmotic pressure, and there remains room for improvement in productivity per cell, etc. Moreover, there is also attempted production of succinic acid using Enterobacter bacteria (WO02008/133131, WO2008/133161).
As for the breeding of such bacteria as described above and in regards to the anaplerotic pathway, the production of non-amino organic acids by fermentation utilizing strains of Escherichia coli, coryneform bacterium, etc., and the like have been reported. Specifically, in these bacteria, phosphoenolpyruvate carboxylase (PEPC) activity or pyruvate carboxylase (PYC) activity is enhanced (Japanese Patent Laid-open Nos. 11-196888 and 11-196887, Applied and Environmental Microbiology (1996), 62, 1808-1810).
As for phosphoenolpyruvate carboxykinase (PEPCK), it is thought that this enzyme generates phosphoenolpyruvic acid from oxalacetic acid by decarboxylation, and it mainly advances the metabolic reactions toward glyconeogenesis (Applied and Environmental Microbiology (1996), 62, 1808-1810, Applied and Environmental Microbiology (1993), 59, 4261-4265). Another type of PEPCK enzyme has been reported that is in equilibrium with the reverse reaction of the reaction described above, that is, the reaction that generates oxalacetic acid from phosphoenolpyruvic acid by carbon dioxide fixation. The presence of this type of PEPCK has been confirmed in some bacteria which produce succinic acid in the presence of high concentrations of carbon dioxide, that is, Mannheimia succiniciproducens, Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, and Selenomonas ruminantium (Applied and Environmental Microbiology (2006), 72, 1939-1948, Applied and Environmental Microbiology (1997), 63, 2273-2280, Applied and Environmental Microbiology (2004), 70, 1238-1241, Microbiology (2001), 147, 681-690). It has also been reported that increasing the activity of PEPCK derived from Actinobacillus succinogenes in Escherichia coli is effective for increasing production of succinic acid (Applied and Environmental Microbiology (2004), 70, 1238-1241). However, this improvement was confirmed only in a PEPC-deficient strain, and the converse has also been reported, in that the increase in the production of succinic acid is NOT observed in a non-deficient strain (Applied and Environmental Microbiology (2004), 70, 1238-1241).
Further, there are also reported production of a non-amino organic acid by fermentation using a strain in which a mutation is introduced into the ptsG gene coding for the membrane binding subunit IICB (Glc) of the glucose phosphotransferase system (PTS) or the ptsG gene is deleted, and the like (for example, Japanese Patent Laid-open Based on PCT Application (KOHYO) No. 2007-535926, Applied and Environmental Microbiology (2001), 67, 148-154, Biotechnology Letters (2006), 28, 89-93). However, it is not reported that a marked effect is attainable on succinic acid fermentation by inactivating the ptsG gene, and further enhancing the activity of PEPCK.